Abstract [en]

Neuronal specification is often seen as a multistep process: earlier regulators confer broad neuronal identity and are followed by combinatorial codes specifying neuronal properties unique to specific subtypes. However, it is still unclear whether early regulators are re-deployed in subtype-specific combinatorial codes, and whether early patterning events act to restrict the developmental potential of postmitotic cells. Here, we use the differential peptidergic fate of two lineage-related peptidergic neurons in the Drosophila ventral nerve cord to show how, in a feedforward mechanism, earlier determinants become critical players in later combinatorial codes. Amongst the progeny of neuroblast 5-6 are two peptidergic neurons: one expresses FMRFamide and the other one expresses Nplp1 and the dopamine receptor DopR. We show the HLH gene collier functions at three different levels to progressively restrict neuronal identity in the 5-6 lineage. At the final step, collier is the critical combinatorial factor that differentiates two partially overlapping combinatorial codes that define FMRFamide versus Nplp1/DopR identity. Misexpression experiments reveal that both codes can activate neuropeptide gene expression in vast numbers of neurons. Despite their partially overlapping composition, we find that the codes are remarkably specific, with each code activating only the proper neuropeptide gene. These results indicate that a limited number of regulators may constitute a potent combinatorial code that dictates unique neuronal cell fate, and that such codes show a surprising disregard for many global instructive cues.

Abstract [en]

The human central nervous system (CNS) contains a daunting number of cells and tremendous cellular diversity. A fundamental challenge of developmental neurobiology is to address the questions of how so many different types of neurons and glia can be generated at the precise time and place, making precisely the right connections. Resolving this issue involves dissecting the elaborate genetic networks that act within neurons and glia, as well as in the neural progenitor cells that generates them, to specify their identities.

My PhD project has involved addressing a number of unresolved issues pertaining to how neural progenitor cells are specified to generate different types of neurons and glial cells in different temporal and spatial domains, and also how these early temporal and spatial cues are integrated to activate late cell fate determinants, which act in post-mitotic neural cells to activate distinct batteries of terminal differentiation genes.

Analyzing the development of a specific Drosophila melanogaster (Drosophila) CNS stem cell – the neuroblast 5-6 (NB5-6) – we have identified several novel mechanisms of cell fate specification in the Drosophila CNS. We find that, within this lineage, the differential specification of a group of sequentially generated neurons – the Ap cluster neurons – is critically dependent upon the simultaneous triggering of two opposing feed-forward loops (FFLs) within the neuroblast. The first FFL involves cell fate determinants and progresses within the post-mitotic neurons to establish a highly specific combinatorial code of regulators, which activates a distinct battery of terminal differentiation genes. The second loop, which progresses in the neuroblast, involves temporal and sub-temporal genes that together oppose the progression of the first FFL. This leads to the establishment of an alternative code of regulators in late-born Ap cluster neurons, whereby alternative cell fates are specified. Furthermore, we find that the generation and specification of the Ap cluster neurons is modulated along the neuraxis by two different mechanisms. In abdominal segments, Hox genes of the Bithorax cluster integrates with Pbx/Meis factors to instruct NB5-6 to leave the cell cycle before the Ap cluster neurons are generated. In brain segments, Ap cluster neuron equivalents are generated, but improperly specified due to the absence of the proper Hox and temporal code. Additionally, in thoracic segments we find that the specification of the Ap cluster neurons is critically dependent upon the integration of the Hox, Pbx/Meis, and the temporal genes, in the activation of the critical cell fate determinant FFL.

We speculate that the developmental principles of (i) feed-forward combinatorial coding; (ii) simultaneously triggered yet opposing feed-forward loops; and (iii) integration of different Hox, Pbx/Meis, and temporal factors, at different axial levels to control inter-segmental differences in lineage progression and specification; might be used widely throughout the animal kingdom to generate cell type diversity in the CNS.

Abstract [en]

The nervous system contains vast numbers of neuronal sub-types, generated at specific time points, in the proper location, and in proper numbers. One of the fundamental issues in neurobiology is to understand the molecular genetic mechanisms that underlie the generation of this daunting neuronal diversity.

To help shed light upon these fundamental questions, my PhD project has addressed the generation and specification of a certain group of neurons, the Ap cluster. This group of four neurons is found only in thoracic segments within the Drosophila melanogaster central nervous system, and consists of three different cell types. Mapping of the neuroblast (stem cell) that generates the Ap cluster neurons, neuroblast 5-6, and the highly restricted appearance of this cluster allowed me to address the following questions: How does NB 5-6 change its temporal competence over time to generate the Ap cluster neurons late in the lineage, and how is temporal competence altered to ensure diversity among the Ap neurons? What are the mechanisms that allow these Ap cluster neurons to emerge only in the thoracic segments?

My studies have helped identify a number of mechanisms acting to specify the Ap cluster neurons. One type of mechanism involves several of different feed-forward loops that play out during NB 5-6 lineage development. These are triggered within the stem cell, where the temporal gene castor activates a number of genes. These castor targets are subsequently involved in several regulatory feed-forward loops, that ultimately result in the unique combinatorial expression of cell fate determinants in the different Ap neurons, which in turn ultimately lead to the activation of unique terminal differentiation genes. In addition, I have identified three different mechanisms by which the NB 5-6 lineage is modulated along the neuroaxis. In the abdomen I find that an early cell cycle exit is initiated by the Bx-C gene members and Pbx/Meis cofactors, which result in the truncation of the NB 5-6 lineage, preventing the Ap cluster neurons from being generated. In thoracic segments Hox, Pbx/Meisand temporal genes act in concert to specify Ap cluster neurons, by integrating with the castor temporal gene. In anterior segments, improper Hox and temporal coding results in a failure to specify bona fide Ap cluster neurons, even though equivalents of Ap cluster neurons are generated.

In summary, my thesis work has helped identify a number of mechanisms acting to specify this unique neuronal sub-type, including: feed-forward combinatorial coding, opposing feed-forward loops and integrated temporal/Hox mediated specification throughout different axial levels. I suggest that these mechanisms may be widely used within the animal kingdom, hence contributing to the great cellular diversity observed within the central nervous system of most animal species.